cerebral preconditioning and ischaemic tolerance

Departments of Neurosurgery, Cell Biology and Physiology, and Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri 63110, USA. e-mail: [email protected]:10.1038/nrn1927

Ischaemic toleranceA condition of transiently increased resistance to ischaemic injury as a result of the activation of endogenous adaptive mechanisms by preconditioning.

PreconditioningPresenting a stressful but non-damaging stimulus to cells, tissues or organisms to promote a transient adaptive response so that injury resulting from subsequent exposure to a harmful stimulus is reduced.

AnoxiaComplete lack of oxygen; in contrast to hypoxia, or low oxygen.

Cerebral preconditioning and ischaemic toleranceJeffrey M. Gidday

Abstract | Adaptation is one of physiology’s fundamental tenets, operating not only at the level of species, as Darwin proposed, but also at the level of tissues, cells, molecules and, perhaps, genes. During recent years, stroke neurobiologists have advanced a considerable body of evidence supporting the hypothesis that, with experimental coaxing, the mammalian brain can adapt to injurious insults such as cerebral ischaemia to promote cell survival in the face of subsequent injury. Establishing this protective phenotype in response to stress depends on a coordinated response at the genomic, molecular, cellular and tissue levels. Here, I summarize our current understanding of how ‘preconditioning’ stimuli trigger a cerebroprotective state known as cerebral ‘ischaemic tolerance’.

The brain’s resistance to ischaemic injury, or ischaemic tolerance, can be transiently augmented by prior expo-sure to a non-injurious preconditioning stimulus. The first in vivo study of cerebral preconditioning documented an acute increase in the capacity of the rat brain for anaerobic glycolysis after brief anoxia, which increased the survival time of the animal following a subsequent exposure to prolonged anoxia1. That the amplitude of an electrically-evoked population spike from hippocampal CA1 neurons was maintained during anoxia if the slice was exposed earlier to brief anoxia led to the proposal in 1986 by Schurr et al.2 that something akin to ischaemic tolerance might exist. The first landmark paper on car-diac preconditioning in dogs by brief coronary ischaemia was published in the same year3. The ability of brief hyperthermia to protect against subsequent focal stroke was documented shortly thereafter4, but was not recog-nized at the time as a type of preconditioning-induced ischaemic tolerance. Rather, it was the dramatic finding that the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells after global ischaemia could be com-pletely prevented if carotid blood flow was briefly inter-rupted 2 days earlier5 that launched the field of ischaemic tolerance research in the brain. Today, a number of robust and reproducible experimental models of cerebral ischae-mic tolerance are recognized (see online Supplementary information S1 (table); for recent reviews, see REFS 6–10). Although rodent and neuronal cell culture models serve as the foundation for the field so far, emerging evidence indicates that ischaemic tolerance is an evolutionarily conserved form of cerebral plasticity that occurs in inver-tebrates and vertebrates, including humans.

Even without preconditioning, resident brain cells naturally respond to ischaemia by mobilizing a host of defences and counter responses to mitigate cell injury and death11 (FIG. 1). However, preconditioning triggers a fundamentally different adaptive response and, in mammals, this response is characterized by at least two distinct time frames of induced tolerance relative to the preconditioning stimulus and the subsequent ischaemia. A short-lasting protective phenotype can be induced within minutes of exposure to precon-ditioning stimuli2,12-14, as a result of changes in ion channel permeabilities, protein phosphorylation and other post-translational modifications; this is known as rapid preconditioning. However, the phenomenon of ischaemic tolerance is best appreciated as one that requires gene activation and de novo protein synthesis (FIG. 1); this ‘classical preconditioning’ requires many hours or even days to become fully manifest. Without another preconditioning stimulus to sustain it, the window for protection also wanes within days. The expression profile of gene activation and repression set in motion by preconditioning is not only tempo-rally specific (see below), but also differs between neurons, glia and endothelial cells. The overall impli-cation is that diverse families of pro-survival genes are activated and, in turn, encode proteins that serve to enhance the brain’s resistance to ischaemia. Protection is achieved by the attenuation of broad categories of injury-inducing mechanisms, including excitotoxic-ity, ion/pH imbalance, oxidative and nitrosative stress, metabolic dysfunction, inflammation and, ultimately, necrotic and apoptotic cell death. Protection is also

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NATURE REVIEWS | NEUROSCIENCE VOLUME 7 | JUNE 2006 | 437

No preconditioning Preconditioning

Ischaemia

Injuriousresponses

Innateprotectiveresponses

Injury phenotype

Preconditioningstimulus

Preconditioning-inducedprotective responses (pre-ischaemia)

Latent cerebroprotectivephenotype

Preconditioning-inducedprotective responses(post-ischaemia)

Ischaemia-tolerant phenotype

Effectors

Transcription factors

Genes

Sensors and transducers

*Response components

Injuriousresponses

Innateprotectiveresponses

*

*

IschaemiaThe condition of reduced or blocked blood flow to a tissue, which, as a result of reduced oxygen and nutrient delivery, can lead to tissue injury.

StrokeA cerebrovascular injury in which blood supply to part of the brain is suddenly interrupted by vessel occlusion (ischaemic stroke) or by vessel wall rupture (haemorrhagic stroke); brain damage and death can rapidly ensue.

Cerebral plasticityThe ability of the brain to reorganize or change both structurally and functionally in response to a challenge, stressor or new experience; this change can be short- or long-lasting, or permanent.

Oxidative and nitrosative stressA potentially helpful or harmful condition that is due to the actions of molecular compounds with reactive oxygen or reactive nitrogen groups, respectively.

manifested by engaging innate survival mechanisms and enhancing endogenous repair processes (for example, the proliferation and mobilization of bone marrow and other stem cells) that lessen the over-all extent of injury and concomitantly facilitate the recovery of brain function.

Broadly speaking, classical preconditioning and the acquisition of the ischaemia-tolerant phenotype involves particular adaptation response cascades to specific stimuli occurring over distinct time frames15,16. Whether the triggering stimulus is preconditioning alone, ischaemia alone, or ischaemia with prior precon-ditioning, each of the cascades can be characterized by gene-dependent responses involving a unique family of sensors, transducers and transcription factors that acti-vate them and the effector proteins that are produced; gene-independent, post-translational modifications of existing proteins also contribute concomitantly to the response (FIG. 1). In brief, preconditioning itself initiates many adaptive reactions in cells and tissues that lead, after some time, to the establishment of what might be

known as a ‘latent’ cerebroprotective phenotype, prim-ing the tissue for the actual injurious ischaemic event. Ischaemia in a preconditioned brain then activates a unique and separate set of adaptive responses. Although changes in the expression of some genes and the modi-fication of some proteins may be shared among these two protective responses, the results of studies carried out so far suggest that their genetic and molecular basis is distinct. In addition, with respect to the response to ischaemic injury, preconditioning does not simply activate competing mechanisms to counteract these injury cascades, but actually serves to genetically ‘repro-gramme’ the response to ischaemia16–18.

In this review I describe the molecular and physi-ological regulation of these survival-promoting mecha-nisms and examine their integration, from the level of subcellular organelles to that of the whole brain. The investigation of endogenous pathways by which the brain protects itself from ischaemia represents a new paradigm for research into stroke — one that holds promise for identifying unique stroke therapeutics.

Figure 1 | Cerebroprotection by preconditioning. This protection requires several stimulus-specific and temporally-defined response cascades that, through a variety of effector molecules, are collectively responsible for establishing the ischaemia-tolerant phenotype. In the non-preconditioned brain, ischaemia-induced injurious responses (red arrows), tempered by innate protective responses that are also activated by ischaemia11, lead to the well-known injury phenotype. However, the ischaemia-tolerant phenotype is the result of both pre- and post-ischaemic protective responses induced by preconditioning, each of which can be considered to be comprised of unique and shared effector cascade response components (see inset). The effectors in these cascades are either produced de novo as a result of changes in gene expression (triggered by distinct sensors, transducers and transcription factors) or are existing proteins that become activated by post-translational modifications. Therefore, even prior to ischaemia, preconditioning establishes a latent cerebroprotective phenotype. When ischaemia ensues, these effects contribute to the overall protection, joining together with other specific protective responses that ischaemia triggers in a ‘reprogrammed’, preconditioned brain. In addition to directly abrogating the injury-promoting cascades (thin red arrow), these responses render the brain more resistant to ischaemic injury and, collectively, serve as the mechanistic foundation of the ischaemia-tolerant phenotype.

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Reactive oxygen speciesHighly reactive compounds containing oxygen with an unpaired electron; at low concentrations they subserve signalling functions, but at higher concentrations they can damage cellular macromolecules.

Inflammatory cytokinesMembers of a group of intercellular signalling molecules, produced by stimulated immune cells and other cells, that trigger and/or amplify inflammatory responses.

CaspasesA family of aspartate-specific cysteine proteases most well known for their involvement in promoting apoptotic cell death, although they may also exhibit apoptosis-independent signalling functions.

Spreading depressionA decrease in neuronal activity evoked by local stimulation of brain tissue leading to a wave of depolarization that spreads slowly across the entire tissue.

Autocrine and paracrineA form of localized signalling in which a cell secretes a given substance that then acts on the same cell, or neighbouring target cells to achieve a biological effect.

NormoxiaNormal (sea level) oxygen levels.

Proteasome pathwayA mechanism whereby proteins are degraded by other proteins; these other proteins often exist as a complex of various proteases.

Preconditioning stimuliDifferences in the intensity, duration, and/or frequency of a particular stress stimulus determine whether that stimulus is too weak to elicit any response, of sufficient magnitude to serve as a preconditioning trigger, or too robust and therefore harmful (for a review, see REF. 8). Therefore, molecules known to cause ischaemic brain injury — such as glutamate, reactive oxygen species, inflammatory cytokines and caspases — might, at the lower levels achieved in response to a particular precondition-ing stimulus, trigger adaptive rather than deleterious responses in resident brain cells19–30.

Although brief cerebral ischaemia, or cerebral hypoxia (for a review, see REF. 10), serve as prototypi-cal preconditioning stimuli, ischaemic tolerance can be induced by exposing animals or cells to diverse types of endogenous and exogenous stimuli that are not necessarily hypoxic or ischaemic in nature (see online Supplementary information S1 (table)). These include spreading depression, hyperoxia and oxidative stress, prolonged hypoperfusion, and, as alluded to above, hyperthermia or heat shock. Therefore, one stressor can promote ‘cross-tolerance’ to another. The sheer variety of stimuli capable of inducing an ischaemia-resistant phenotype in the brain indicates that the signalling pathways activated by these different triggers converge downstream on some common, fundamental mecha-nisms that ultimately account for the protection (see below). Many exogenously delivered chemical precon-ditioning agents (for example, inflammatory cytokines, anaesthetics and metabolic inhibitors) can also induce ischaemic tolerance, raising the hope that in the future it will be possible to pharmacologically activate these distal pathways in the human brain. Moreover, although they have yet to be identified, the molecular triggers capable of establishing ischaemic tolerance can be blood-borne: brief ischaemia of skeletal muscle promotes ‘remote’ tolerance against cerebral ischaemia30, as does physical exercise31,32.

CNS-specific antigens such as myelin basic pro-tein33, myelin oligodendrocyte glycoprotein34 and even E-selectin35 can also serve as preconditioning stimuli when administered systemically. This results from a type of immunological tolerance, known as bystander sup-pression33, whereby the subsequent release of the same antigens in response to ischaemia-induced blood–brain barrier damage triggers the recruitment of regulatory T cells that produce anti-inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth fac-tor-β (TGFβ). Therefore, if a particular preconditioning challenge exposes CNS antigens to the blood, secondary to a transient opening of the blood–brain barrier, it is possible that bystander suppression could contribute to the ischaemia-tolerant phenotype induced by that stimulus.

Sensors and transducersTo induce tolerance, the preconditioning stimulus must be recognized by molecular sensors as a sign of something potentially much more severe to come. So far, numerous types of sensor have been identified,

including neurotransmitter, neuromodulator, cytokine and toll-like receptors36, as well as ion channels and redox-sensitive enzymes. In turn, these sensors acti-vate transduction pathways that initiate the adaptive response. Although dependent in part on the nature of the preconditioning stimulus, members of these transduction pathways for which there is strong gen-eral support include mitogen-activated protein kinases (MAPKs) and their phosphorylated Ras, Raf, MEK and ERK subfamilies6,37,38, mitochondrial ATP-sensi-tive K+ (KATP) channels39,40, Akt (also known as protein kinase B)41–43 and the protein kinase C-ε isoform44.

The possibility that the nitric oxide-based adaptive response to hypoxia in Drosophila45 is evolutionarily conserved suggests that this multifunctional modulator might be a logical choice as an autocrine and paracrine mediator of preconditioning stress. Indeed, pharmaco-logical and genetic evidence supporting the involvement of nitric oxide (derived from the endothelial30,43,46–49, neu-ronal37,48 and inducible isoforms of nitric oxide synthase (NOS)50,51) in the transduction process is continuing to mount. Given the redox sensitivity of many kinases and transcription factors, reactive oxygen species might also serve as transducers28,29,52. Adenosine, another prototypi-cal paracrine mediator and ‘retaliatory metabolite’, the production of which is linked to ATP degradation, seems integral to tolerance induction in some models39,40,53,54. Finally, caspases might be essential induction catalysts, given that cyclic AMP responsive element-binding protein (CREB), the p50 and p65 subunits of nuclear factor-κB (NF-κB), and protein kinase C and other kinases are caspase substrates26,55. Notably, some of the aforementioned molecular transducers and signalling intermediates also serve as post-ischaemic effectors of the ischaemia-tolerant phenotype8 (see below).

Transcription factors. Preconditioning-activated sig-nalling pathways converge to induce post-translational modifications of existing proteins and/or to activate transcription factors that drive the genomic response. Several transcription factors are known to be sensitive to regulation by hypoxia/ischaemia and probably par-ticipate in this response, including activating protein 1 (AP1)56, CREB57–59, NF-κB52,60, early growth response 1 and the redox-regulated transcriptional activator SP1(REF.61). However, the hypoxia-inducible factor (HIF) isoforms have garnered the most experimental support so far with respect to mediating the transac-tivation of adaptive, pro-survival genes, particularly those involved in glucose metabolism and angiogen-esis10,62–64. Transcriptional regulation of the genome by HIF is similar in Drosophila, C. elegans and mammals. In mammals, the HIF1α isoform is hydroxylated dur-ing normoxia by an oxygen- and iron-dependent prolyl hydroxylase, allowing interaction between HIF1α and an ubiquitin ligase that targets HIF1α for degradation by the proteasome pathway. However, hypoxia renders the hydroxylase enzyme nonfunctional, and HIF1α, no longer able to associate with the ligase in its non-hydroxylated form, then enters the nucleus, dimerizes with HIF1β, and promotes the transcription of genes

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that enhance hypoxic resistance. Prolyl hydroxylase inhibitors such as deferroxamine, cobalt chloride and other ‘HIF-mimetics’ are therefore attracting attention as potential preconditioning therapeutics (for reviews, see REFS 10,65). The HIF2α isoform is regulated in a similar way. It exists primarily in endothelial cells and, although it seems to be important in embryonic vasculogenesis, it does not seem to be induced in response to hypoxia in the neonate brain15 as it is in the adult brain66. Details regarding transcriptional regulation by HIF2α are still unclear; although it may co-transactivate some genes with HIF1α, through the influence of kinases and other regulators, HIF2α might be more active on the promot-ers of endothelial cell-specific survival genes67 associated with angiogenesis, vascular remodelling, and endothelial cell homeostasis.

EffectorsPrimarily through recent oligonucleotide-based DNA microarray investigations15–18,68 and novel gene identi-fication methods69, the transcriptome of the ischaemia-tolerant brain is gaining definition. Several themes are emerging from this work. First, genes representing many larger ‘families’ participate in the response — given the robustness of the protection in most tolerance models, this was not unexpected. Many share common promoter sequences that are responsive to preconditioning-acti-vated transcription factors. On balance, genes function-ally related to the cell cycle, metabolism, inflammation, excitotoxicity, ion homeostasis, signal transduction and so on are differentially expressed in response to precon-ditioning15–18,68.

Second, genes activated by preconditioning stimuli are often quite distinct from those associated with ischaemia alone; similarly, the genomic expression pat-tern in response to ischaemia is unique in a precondi-tioned animal, and differs considerably from the pattern activated by either preconditioning or ischaemia in a non-preconditioned animal16.

Third, although important and interesting from a cell survival standpoint, the genomic response is not simply one of activation of normally quiescent survival genes. Rather, gene repression also occurs, and, in fact, dominates the overall response to ischaemia in a preconditioned brain16,18. Finally, changes in gene tran-scription after preconditioning, or after ischaemia in a preconditioned brain, have distinct temporal profiles. For example, following preconditioning, some genes are expressed or repressed within minutes or hours (adeno-sine A2a receptor and vascular endothelial growth fac-tor (VEGF)15,18), whereas others are affected days later (β-actin, serine/threonine protein kinase, arachidonate 12-lipoxygenase, calretinin, the S100A5 calcium-bind-ing protein, dihydropyrimidine dehydrogenase and the zinc transporter ZnT1 (REFS 15–18). Some change transiently (CCAAT/enhancer-binding protein-related transcription factor, adenosine A2a receptor18 and met-allothionein II (REFS 15,17,18)), whereas others change for a protracted time (heat shock proteins, BCL2, p38-MAPK, TGFβ1, Iκ-Bα, glial fibrillary acidic protein and β-tubulin15,17,18).

The generation of these rapidly expanding genomic datasets must ultimately be coupled to the identifica-tion of translational expression patterns that define the ischaemia-tolerant proteome, as not all of the changes in mRNA levels identified by the previously mentioned microarray studies will be realized as proportional changes in protein levels. Findings so far indicate that cellular-level effectors of ischaemic tolerance are ubiq-uitous, and include structural and functional proteins of the cell membrane, cytoskeleton, mitochondria, and other organelles that have widespread actions on cel-lular metabolism. At least three unique time windows of phenotypic, effector mechanism expression — that is, before, during and after ischaemia — are worthy of closer examination.

The first is the period of time that follows precon-ditioning but is prior to the lethal ischaemic insult. At the cellular level, significant alterations in protein composition (such as elevations in the concentrations of various stress proteins, kinases and phosphatases, transcription factors, metabolic enzymes, transport and structural proteins, trophic factors and plasticity-related molecules, and cell cycle/apoptosis-related proteins) are underway or established prior to ischaemia. These new protein signatures are representative of a tissue that is prepared to resist the threat of an impending ischaemic event (FIG. 1).

The second window of effector mechanism expres-sion relates to the period of ischaemia itself, such that an identical insult is experienced as less severe in a precon-ditioned brain compared with a naive brain. In part, this occurs through increased inhibitory neurotransmitter levels, lower intra-ischaemic levels of extracellular gluta-mate, reductions in calcium influx secondary to altered AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole pro-pionic acid) receptor subunit compositions, adenylation states, and desensitization status70–73. Whether levels of high-energy phosphate reserves and glycolytic and oxi-dative intermediates are altered during the ischaemic interval is controversial and may be age- and model-dependent74–76.

Pathophysiological events occurring during post-ischaemic reperfusion are also positively modulated in ischaemic tolerance. Effector mechanisms acting during this time window broadly serve to stabilize the cell’s energy and protein metabolism, ameliorate the actions of glutamate, reactive oxygen and nitrogen species and other injurious mediators, and reduce post-ischaemic inflammation. Tolerance mechanisms working at the level of endoplasmic reticulum func-tion result in improved rates of recovery of neuronal protein synthesis75,77; specifically, better preservation of the reinitiation and elongation steps of transcription, and increased levels of the chaperone glucose regulated protein 78 kDa (GRP78) are realized78,79. In addition, post-ischaemic protein aggregation, redistribution and ubiquitin-conjugation80 are reduced. The rate of repair of oxidative DNA damage is also enhanced81,82. Overall, many facets of ischaemic mitochondrial dysfunction, including changes in the redox activity of the respira-tory chain components, oxidative phosphorylation

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Decoy receptorsSoluble or cell-surface-binding proteins that bind the ligand with high affinity and specificity, but do not induce a biological response; used in immunological regulation.

deficits83, calcium overload and the initiation of apopto-sis (see below), are abrogated in ischaemic tolerance84,85. Compared with the naive brain, the preconditioned brain also shows post-ischaemic increases in the Mn and CuZn isoforms of superoxide dismutase86,87, glutathione peroxidase and glutathione reductase88, uric acid89, and haeme oxygenase-1 (REF. 87), which enhance the tissue’s free radical scavenging capabilities. Reductions in pro-inflammatory cytokine synthesis90, and an upregulation of other feedback inhibitors of inflammation, including decoy receptors and intracellular signalling inhibitors36, contribute to the promotion of an anti-inflammatory phenotype following ischaemia.

Programmed cell survivalCounterbalancing the well-established programmed cell death pathways activated by ischaemia are the less well-understood ‘programmed cell survival’ pathways. These schemes might represent two sides of the same ischaemic tolerance coin (TABLE 1). However, we know relatively little about the organelle- and cell-specific molecular underpinnings of these survival pathways. Although apoptotic cell death, as an endpoint, is reduced in ischaemic tolerance91–93, so far only changes in rec-ognized apoptotic mediators have been measured to explain this protective effect. For example, we know that preconditioning-induced, CREB-mediated increases in the synthesis of the anti-apoptotic proteins BCL2 (REFS 58,59,91,94,95) and BCL-XL (REF. 95) occur, as well as stabilization of the mitochondrial membrane poten-tial95, decreases in mitochondrial cytochrome c96 and

SMAC/DIABLO release97, and reductions in caspase 3 synthesis98 and p53 activation99. We do not yet know if the production of members of the caspase-neutralizing inhibitors of apoptosis (IAP) family, the interference with the mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF), or the stabilization of intramitochondrial redox and membrane potential by uncoupling proteins100 take place. In addition, the ability of preconditioning to reduce apoptotic cell death is likely to involve groups of as yet unidentified, evolutionarily conserved proteins that have direct and unique cell sur-vival functions, and is not dependent on the inhibition of existing pro-apoptotic pathways alone.

The survival-promoting effects of some endogenous molecules, such as the endoplasmic reticulum-associ-ated chaperone ORP150 (REF. 101) and serum response factor102, as well as survival genes such as alivin 1 (REF.

103), are now being identified, but their role in ischaemic tolerance remains unknown. Other survival-promoting effector proteins, such as those of the neurotrophin fam-ily, have been implicated directly in ischaemic tolerance. For example, preconditioning-induced increases in neu-rotrophin ‘tone’ are one strategy that the tolerant brain uses to derive the various trophic benefits from nerve growth factor104, brain-derived neurotrophic factor (BDNF)104,105, basic fibroblast growth factor106, insulin-like growth factor107 and the neuregulins108. Several stud-ies have now identified Akt, which is phosphorylated by phosphotidylinositol-3-kinase, as crucial to establishing the tolerant phenotype, secondary to an Akt-mediated phosphorylation of mixed lineage kinase 3 (REF. 109),

Table 1 | Molecular mediators of programmed cell death and programmed cell survival

Cellular localization or activity

Programmed cell death Programmed cell survival

Membrane receptors TNFα; FasL; DR3/4/5; adaptor proteins (TRADD, FADD)

Adenosine A1; metabotropic glutamate; P2Y2; dopamine D2; GCSF; PPARα/β/δ; neurotrophin; TNFα

Signal transduction Caspases; ASK1; GSK3β; neuronal NOS Raf, MEK and ERK kinases; PI3K/pAkt; JAK2; STAT5; PKCε; ceramide; endothelial NOS; MLK3

Transcription factors NF-κB; JNK; HIF; FOXO CREB; SP1; HIF; NF-κB; AP1; SRF; FOXO; MEF2

Mitochondrial effectors BAX, BAD, BID, BIM; loss of mitochondrial membrane potential; release of cytochrome c, SMAC/DIABLO and AIF; production of ROS

BCL2; BCL-XL; BCL-W; stabilization of mitochondrial permeability pore; inhibition of cytochrome c and SMAC/DIABLO release; inhibition of AIF translocation; elaboration of thioredoxin; uncoupling protein 2

Nuclear effectors PARP; p53; c-jun; endonucleases; pro-apoptotic genes

IAPs (XIAPs, survivin); DNA repair enzymes; survival genes

Miscellaneous Reduction in caspase activation; endoplasmic reticulum stabilization (ORP150); increases in trophic factors (NGF, BDNF, IGF1, bFGF); synthesis of heat shock proteins and other molecular chaperones; post-translational modifications; production of VEGF, erythropoietin and adenosine

Inhibition of programmed cell death and augmentation of programmed cell survival contribute to preconditioning-induced ischaemic tolerance. AIF, apoptosis-inducing factor; AP1, activator protein 1; ASK1, apoptosis signal-regulating kinase 1; BAD, BCL-associated death promoter; BAX, BCL2-associated protein X; BCL2, B-cell leukaemia/lymphoma 2; BCL-W, BCL2-like protein 2; BCL-XL, BCL2-like protein 1; BID, BH3-interacting domain death agonist; BIM, BCL2-interacting mediator of cell death; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CREB, cyclic AMP responsive element-binding protein; DR, death receptor; ERK, extracellular signal-regulated kinase; FADD, Fas-associated protein with death domain; FasL, Fas ligand; FOXO, forkhead family of transcription factors; GCSF, granulocyte colony-stimulating factor; GSK3β, glycogen synthase kinase 3β; HIF, hypoxia-inducible factor; IAP, inhibitor of apoptosis protein; IGF1, insulin-like growth factor 1; JAK2, Janus tyrosine kinase 2; JNK, c-jun amino terminal kinase; MEF2, myocyte-enhancing factor 2; MEK, mitogen-activated protein kinase kinase; MLK3, mixed lineage kinase 3; NF-κB, nuclear factor-κB; NGF, nerve growth factor; NOS, nitric oxide synthase; ORP150, oxygen-regulated protein 150; P2Y2, purinergic receptor P2Y; p53, tumour suppressor p53; pAkt, phosphorylated Akt; PI3K, phosphotidyl inositol-3-phosphate; PARP, poly(ADP-ribose)polymerase; PKCε, ε isoform of protein kinase C; PPAR, peroxisome proliferator activator; Raf, small G-protein; ROS, reactive oxygen species; SMAC/DIABLO, second mitochondria-derived activator of caspase/direct IAP binding protein with low pI; SP1, specificity protein 1; SRF, serum response factor; STAT5, signal transducer and activator of transduction 5; TNFα, tumour necrosis factor-α; TRADD, TNF receptor-1-associated death domain protein; VEGF, vascular endothelial growth factor; XIAP, X-linked IAP.

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Neurovascular unitA practical construct consisting of brain endothelium, astrocytes and microglia, neurons, and the extracellular matrix, and the dynamic interactions that occur between them in health and disease.

endothelial NOS43, the IAP protein survivin110 and, probably, many other cytoprotective effectors. VEGF, a HIF-driven gene, exhibits several distinct neurotrophic and neuroprotective effects relevant to post-ischaemic angiogenesis and neurogenesis111. Evidence is rapidly accumulating that erythropoietin, another HIF-regulated protein, participates as an endogenous pro-survival fac-tor in ischaemic tolerance (BOX 1), and illustrates how the identification of endogenous survival-promoting molecules and their mechanisms of action may provide therapeutic targets for stroke.

Integrating the responseOn the basis of studies in in vitro models, neurons, glia and endothelial cells can be rendered ischaemia-toler-ant by preconditioning (see online Supplementary information S1 (table)), underscoring the capacity for intrinsic cellular-level tolerance without the need for communication between cell types, the involvement of the surrounding macro- and microvascular networks, or the participation of systemically-acting mediators. Nevertheless, achieving optimal levels of tolerance in vivo requires that the unique phenotypic response of each of these cells, as well as those invoked in resident immune cells, vascular smooth muscle cells and circu-lating leukocytes, are coordinated and assimilated to establish a tangible survival advantage across the whole tissue at risk. For example, co-culture studies show that the synthesis and release of erythropoietin112 and other

molecules (see below) by astrocytes contribute impor-tantly to the preconditioning-induced protection of neighbouring neurons. This section gives consideration to the adaptive responses that occur at the level of the neurovascular unit, which, when integrated temporally and spatially, provide robust protection in response to in vivo preconditioning.

Vascular ischaemic tolerance. Although neurons are inherently assumed to be the cellular target of cerebral preconditioning, ischaemic tolerance occurring at the level of endothelial and smooth muscle cells (and manifested across several levels of vascular organization) contributes importantly to neuronal protection. At the tissue level, absolute regional blood flow, either before113 or during113–115 ischaemia, does not seem to be altered by preconditioning; cerebral metabolism also remains unchanged25,50,116. However, reductions in absolute114,117 and relative118 blood flow after permanent focal stroke are lessened by prior preconditioning, concomitant with an increase in the number of histologically-identified pat-ent microvessels114. Prior preconditioning also reverses the hypoperfusion that occurs early during reperfusion following global ischaemia119. With respect to vascular reactivity, post-ischaemic endothelium-dependent vasodilation is better preserved in the preconditioned brain120. Studies have yet to show that these improve-ments in post-ischaemic blood flow and reactivity contribute to the reduction in lesion magnitude that is associated with ischaemic tolerance.

Vasculoprotective tolerance mechanisms operating at the microvascular level account, in part, for the anti-inflammatory phenotype that characterizes ischaemic tolerance. For example, reductions in vasogenic oedema are evident in newborn121 and adult30,122,123 models of ischaemic tolerance, but how prior preconditioning enhances or maintains blood–brain barrier integrity in the face of ischaemia remains unclarified. Reductions in leukocyte–endothelial interactions and diapedesis of neutrophils and other leukocytes may be one explana-tion, as found in rats after exercise preconditioning32 and in mice after lipopolysaccharide precondition-ing124, although investigations to establish a causal link between these changes and improved barrier function are required. Microarray studies16,17, in vivo immunohistochemistry32 and immunoblotting in a cerebral endothelial cell culture model of ischaemic tolerance125 indicate that a reduction in the expres-sion of members of the selectin and immunoglobulin adhesion molecule families following ischaemia may reduce leukocyte–endothelial rolling and adherence, and thereby attenuate infiltration. Moreover, even the proteome of circulating leukocytes is affected by preconditioning, as shown by transient suppression of the leukocyte genes encoding adhesion and migration, chemotaxis and cytokine synthesis after brief ischae-mia in the human forearm126; indeed, blood monocytes show less activation after ischaemia in a preconditioned brain124. Such preservation of cell–cell homeostasis at the endothelial–blood interface might result from the anti-inflammatory effects of endothelially-derived

Box 1 | Erythropoietin: translational research success in the making?

Originally identified as a kidney-derived glycoprotein hormone responsible for erythroid progenitor cell proliferation, erythropoietin is now recognized as a pleiotropic cytoprotective cytokine and a prominent hypoxia-inducible factor (HIF) gene target147. Although exogenous erythropoietin is neuroprotective in stroke148–150, preconditioning with hypoxia63,151 or deferroxamine152 increases endogenous cerebral erythropoietin mRNA and protein levels. Even erythropoietin itself, administered 24 h before cerebral ischaemia, promotes tolerance153,154. Blockade of the protective effects of preconditioning by soluble erythropoietin receptors or erythropoietin antisense112,151,155 provides causal evidence for erythropoietin’s participation in ischaemic tolerance.

The beneficial cytoprotective effects of erythropoietin are mediated by erythropoietin receptors (EPORs, yet to be subclassified) located on neurons, astrocytes, microglia and endothelial cells153. Lumenally-oriented EPORs on endothelial cells149 may transcytose exogenously delivered erythropoietin from blood to brain; in particular, to the EPORs on the astrocytic end-feet that ensheath cerebral capillaries153, to initiate astrocyte-mediated protective mechanisms129.

Mechanistically, erythropoietin-mediated protection involves a multidimensional array of actions, triggered by the phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 5 (STAT5), Akt (also known as protein kinase B) and other signalling complexes that ultimately reduce apoptosis , reactive oxygen species production and inflammation156. Erythropoietin also has pro-angiogenic effects157 and regulates stem cell trafficking and neurogenesis158.

As millions of patients with anaemia tolerate erythropoietin therapy, its safety profile supports the use of short-term erythropoietin application for acute stroke. In the first Phase I/II clinical trial, high-dose recombinant erythropoietin administered within 8 h of symptom onset significantly improved early clinical outcome measures159; a Phase III, multicentre trial is now underway in Germany. Efforts to create a designer cytokine with fewer erythropoietic and prothrombotic effects relative to its non-haematopoietic effects that crosses the blood–brain barrier have proved successful in several different animal models of neurological disease160,161, and may be the preferred construct for long-term preconditioning of patients at high risk for stroke.

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Epoxy eicosatrienoic acidsEpoxides of arachidonic acid generated by cytochrome P450 epoxygenases that have various regulatory actions.

nitric oxide, secondary to a preconditioning-induced, Akt-dependent phosphorylation of endothelial NOS43,118 that enhances its activity47,118. Nothing is known about platelet dynamics at the endothelial–blood interface: although the blood of preconditioned mice has a prolonged coagulation time, this is not as a result of a reduction in platelet number, but might be secondary to preconditioning-induced increases in cyclooxygen-ase 1 and prostacyclin synthase16. If this were the case, then the resultant reduction in ischaemic microvascular thrombus potential would provide another advantage to the preconditioned brain.

A fully functioning cerebrovascular endothelium is also crucial to achieving optimal post-ischaemic angio-genesis and microvascular remodelling. The activation of endothelial NF-κB127, the production of and responsive-ness to VEGF42, and the induction of distinct endothelial cell-specific anti-apoptotic response networks128 may be just a few of the means by which prior preconditioning supports these neovascular responses that are so essential to long-term recovery. The selectively enhanced recruit-ment of endothelial progenitor cells to ischaemic brain regions would also be a beneficial feature of ischaemic tolerance, but this possibility has not yet been experi-mentally addressed.

Glial ischaemic tolerance. Resident astrocytes and microglia, like vascular cells, also respond uniquely to preconditioning stimuli, and such responses are cru-cial to the foundation of the overall cerebroprotective phenotype (for a review, see REF. 129). Enhancements in astrocytic ion buffering, the transfer of energy substrates and neurotransmitter metabolites to neurons130, func-

tional and structural blood–brain barrier support, free radical scavenging, glycogen storage and erythropoietin production112 are some of the ways in which astrocytes are likely to enhance the ischaemic resistance of neigh-bouring neurons. The generation by preconditioned astrocytes of anti-inflammatory cytokines such as IL-10, heat shock proteins (for example, HSP27 and HSP32), and trophic factors such as TGFβ, BDNF, GDNF (glial-cell-derived neurotrophic factor) and VEGF might also contribute. Recently, evidence for the astrocytic production of epoxy eicosatrienoic acids secondary to a preconditioning-induced, HIF1α-dependent upregula-tion of the arachidonic acid epoxygenase P450 2C11 was advanced64. Morphologically, both astrocytes and micro-glia are transiently activated in response to precondi-tioning131,132, which may be a reflection of the functional responses listed above. Conversely, the well-recognized, more robust activation of astrocytes and microglia/mac-rophages that is triggered by ischaemia is attenuated in previously preconditioned brains124,131, which may be secondary to a repression of NF-κB-mediated transcrip-tion of inflammatory mediators133.

Mechanistic parallels of noteOur understanding of the mechanisms by which pre-conditioning protects the brain will be facilitated by continued investigation of the mechanisms innate to the fetal brain (BOX 2) and the brain of the high-alti-tude native, both of which are able to function in low oxygen environments. Hypoxia- and anoxia-tolerant lower vertebrates and hibernating mammals also hold clues to ischaemic tolerance (see online Supplementary information S2 (table)); many survival responses in these animals are categorically similar, if not identical, to those used for protection in mammalian ischaemic tolerance. In vertebrates, adaptations to oxygen insuf-ficiency are not found in every species in a genus, and therefore are not simply the phylogenetic consequence of some lower level of organization. This suggests the presence of distinct adaptive responses that allow toler-ance to pure anoxia for days in some species and, under more hypothermic conditions, for weeks in others. The primary adaptive response invoked by anoxia-tolerant animals is not to switch to anaerobic metabolism and/or use alternative substrates (a distant second choice), but rather to induce a state of extremely low energy production and utilization (for a review, see REF. 134). In brief, we know that by reducing protein synthesis (metabolic arrest), and by stabilizing membrane func-tion secondary to decreases in ion permeability and ion pumping (channel arrest), neurons can achieve a robust hypometabolic state. Other phenotypic features of anoxia-tolerant species include the avoidance of anoxic depolarization through the elevation of adenosine and inhibitory transmitter levels (and their receptors), more efficient clearance of extracellular glutamate, the storage of large amounts of glycogen, and the augmentation of glutathione peroxidase and ascorbate levels to handle oxidative stress — all complemented at the extracerebral level by circulatory redistribution and bradycardia (for a review, see REF. 135). The cerebral blood flow in the

Box 2 | Endogenous tolerance in the neonate

Compared with adults of the same species, the intrinsic ability of the fetus and newborn to better tolerate hypoxia/ischaemia has long been recognized. Although this advantage is not surprising given the hypoxic intra-uterine environment, elucidating the mechanistic basis of this resistance at the level of the CNS might provide clues as to how preconditioning protects the adult brain. Reductions in oxygen demand are known to underlie the ability of the neonate brain to resist low oxygen tension. This hypometabolic state is achieved by many of the same fundamental alterations that characterize anoxia-tolerant species, including: lower rates of resting glucose metabolism; increases in glucose transport, glycolytic enzymes and glycogen stores; lower densities of NMDA (N-methyl-d-aspartate) channels and channel distribution patterns that reduce overall neuronal excitability and delay depolarization; and other adaptations that slow the rate of high-energy phosphate depletion and maximize ATP homeostasis during and after ischaemia74,76,135,162.

Several models of neonatal ischaemic tolerance have been published, as well as a recent review9. The extent of protection afforded by hypoxic preconditioning in the neonatal rat is exceptionally robust46,74, as is that resulting from hyperthermic121,163 and xenon58 preconditioning. Hypoxic preconditioning in the prenatal period is also effective for reducing hypoxic-ischaemic injury after birth92. It is not clear whether the neonatal brain is already ‘primed’ for preconditioning, and/or if preconditioning augments survival mechanisms already constitutively active in the hypoxia-adapted brain, but the genomic basis of ischaemic tolerance in neonates probably involves many distinct, age-dependent responses. For example, given the propensity for ischaemic neuronal death by apoptosis in the neonate, strong and potentially unique anti-apoptotic mechanisms must underlie ischaemic tolerance at this age58,92,93,107. Conversely, other molecular players and transcription factors involved in the induction phase of preconditioning in the neonatal brain38,46,62,107,164 are not unique to this age group.

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ArousalThe intentional act of periodic brief awakening characteristic of hibernating animals, characterized by distinct physiological changes and states of activity, depending on species.

Translational researchThe process of taking results from the laboratory and translating them into therapies for clinical use.

anoxia-tolerant mammalian brain is maintained during anoxia onset, but thereafter is significantly reduced in parallel with metabolic rate.

Active changes also accompany hibernation: metabo-lism and blood flow are lowered to levels less than 10% of baseline136, body temperature and white cell counts are reduced, and stress kinases and heat shock proteins are activated. Unique lipid and protein sequestration pat-terns have been noted in neuronal endoplasmic reticu-lum137. The mechanisms by which hibernating animals handle the rapid increase in cerebral blood flow and severe oxidative stress during arousal are poorly under-stood, but it is possible that they could be applied in a translational way to meet the similar challenges faced by the human brain during post-ischaemic reperfusion.

Insights into ischaemic tolerance may also be forth-coming from studies of the high-altitude natives of the Andes and Himalayas. Positron emission tomography (PET) studies of the Andean Quechuas have revealed systematically lower region-by-region rates of cerebral glucose utilization compared with lowlanders138. This is consistent with the idea that the same hypometabolic strategy shown by the CNS of anoxia-tolerant animals is used by the human brain. In addition, it is worth not-ing that although the adaptations required by native lowlanders to withstand the hypoxia of high-altitude mountaineering may take weeks or months to establish, the resultant ability to maintain relatively unimpaired cognitive function at extreme altitude indicates that even those brain neurons most vulnerable to hypoxia have the endogenous capacity to adapt to, and success-fully withstand, severe stress.

Current questions and future studiesDespite coverage by ~450 publications since 1991, the field of cerebral preconditioning and ischaemic tolerance is still relatively immature. The same can be said, in gen-eral, of the so-called physiological ‘stress response’, which continues to challenge our understanding. Hundreds of unanswered questions result from the significant gaps that still exist in our knowledge base regarding ischaemic tolerance mechanisms. The few controversies surround-ing particular results published so far are not unexpected in an emerging discipline such as this, not to mention one based on so many different experimental models. In addition to the need to develop well-characterized mod-els of ischaemic tolerance in higher mammals, at least three basic issues regarding preconditioning efficacy in rodents still require more thorough documentation; these issues arise from the historical reliance on the use of either histological or morphological endpoints, or biochemical indices to document protection shortly after the ischaemic insult. First, it is imperative to thoroughly document whether the protection afforded by precondi-tioning is long-lasting and if injury is truly prevented, or whether preconditioning simply delays the development of infarction. Although a few studies have confirmed that sustained protection remains 2–4 weeks after cerebral ischaemia in neonatal and adult models58,74,105,118, oth-ers have shown progressive cell loss at more protracted recovery times of 30 days post-stroke139. Although these outcome measures probably depend on the characteristics of the model under study, the intuitive qualitative predic-tion would be that tolerance is likely to be maximized in magnitude and duration (that is, permanent protection) when the preconditioning stimulus is optimized, and the severity of the subsequent ischaemic insult is not overwhelming. This was borne out experimentally in a study of global ischaemia in rats, in which sustained hippocampal CA1 protection was confirmed at 1 month following ischaemia under ‘idealized’ preconditioning and ischaemia regimens140. Regardless of this, additional work addressing this issue across various outcome measures is needed. A second issue is the functional documentation of ischaemic tolerance, both electrophysiologically and at the neurocognitive, sensorimotor and behavioural levels. In one study, preconditioned animals showed improved orientation and memory function relative to their naive counterparts141. However, another study measured func-tional deficits in tolerant animals even though, based on neuronal morphology, the tissue appeared ischaemia-tolerant142. It is also imperative to document that effective preconditioning stimuli are truly non-injurious; again, not only at histological levels but across more sensitive behavioural metrics as well143. Such studies have impor-tant therapeutic implications with respect to addressing the safety of different preconditioning regimens and their respective side effects. Studies designed to address the aforementioned issues will strengthen the experimental foundation for ischaemic tolerance and, in turn, promote translational research opportunities to further explore its clinical applicability (BOX 3).

Several time-dependent features of preconditioning and ischaemic tolerance deserve further exploration. For

Box 3 | Ischaemic tolerance in the human brain: therapeutic potential?

So far, clinical trials of more than 50 compounds for acute ischaemic stroke have failed. Only thrombolysis-based therapy is currently approved for use in the United States, but treatment is limited to a small fraction of stroke patients meeting specific inclusion criteria. Most drugs that have advanced to clinical trials have been designed to block or limit the key molecular events identified in the laboratory as contributing to neuronal injury. Given the magnitude and reproducibility of cerebroprotection against ischaemia achieved in many diverse models of preconditioning, targeting mechanisms of innate cytoprotection present another strategy for drug development. That experimental studies have successfully used exogenously applied agents to activate dormant neuroprotective genes and cell survival pathways to achieve ischaemic tolerance further underscores the feasibility of identifying pharmacological approaches for increasing endogenous ischaemic resistance; for example, the use of prolyl hydroxlase inhibitors to stabilize HIF1α65 and erythropoietin-based treatment. Patients at high risk for stroke might derive benefit from prophylactic treatment and, given the protracted therapeutic window over which ischaemic brain injury occurs, so might patients presenting shortly after stroke.

Ischaemic tolerance may already occur naturally in humans, in the form of short episodes of ischaemia without infarction, known as transient ischaemic attacks (TIAs). Three retrospective analyses of prospectively collected data165–167 show, by several metrics, a reduced severity of stroke with antecedent ipsilateral TIA. But the number of investigations to date is limited, sample sizes are relatively small, and the clinical status of the patients is likely to be non-uniform. However, the results of large clinical studies support the parallel contention that, in myocardial ischaemia, prior angina is protective. Given the breadth and depth of research on myocardial ischaemic tolerance (with ~3500 papers published on this topic since 1986), preconditioning is much closer to becoming a routine clinical treatment for patients requiring ischaemia-inducing procedures such as coronary angioplasty and coronary artery bypass grafting145.

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example, almost every experimental model used so far has been based on a single preconditioning stimulus and the transient period of either rapid or classical ischaemic tolerance that it elicits. However, the more chronic adap-tations shown by hibernating and diving mammals, and the acclimatization to hypoxia that occurs in humans with extended stays at high altitude, suggest that sus-tained augmentations in resistance to hypoxia-ischaemia can be realized in the brain. Is it possible that repeti-tive presentations of particular preconditioning stimuli could promote significantly longer-lasting changes in the neuroprotective phenotype? Can the brain exhibit a type of sustained adaptive plasticity akin to the idea of chronic ischaemic tolerance? The therapeutic implica-tions of such a finding for patients at a high risk of stroke would be compelling.

Another example is the provocative finding from recent myocardial preconditioning studies that brief, transient interruptions in blood flow during early reperfusion, termed ‘postconditioning’, reduce ischaemic injury to levels similar to that achieved with precondi-tioning144. Moreover, as the beneficial effects of precondi-tioning and postconditioning do not seem to be additive, then perhaps some preconditioning mechanisms may be suitable for activating after ischaemia — a finding that has obvious clinical ramifications145. With the exception

of a recent report showing that lipopolysaccharide, a well-established preconditioning agent47,86,114,118,120,124,146, reduces leukocyte recruitment when given after ischae-mia146, whether this variation on reperfusion injury therapy would also trigger rapid ischaemic tolerance pathways in the brain that are uniquely different from those that underlie other efficacious post-ischaemic therapies in experimental stroke remains unexplored.

ConclusionsStudies of preconditioning and ischaemic tolerance are currently being published at what appears to be a near exponential rate, which should help to advance our understanding of its mechanistic basis and, in turn, its clinical potential. The endogenous survival mechanisms activated in response to preconditioning do not depend on differences in drug pharmacokinetics or adminis-tration protocols that can confound the translation of neuroprotective strategies from rodents to humans. Therefore, the identification of intrinsic cell survival pathways should provide more direct opportunities for translational neuroprotection trials. Although primarily focused on stroke at present, we might find that the innate regulatory schemes that underlie tolerance to ischaemia are also applicable to protecting the brain from other acute and chronic neurodegenerative disorders.

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AcknowledgmentsDue to space limitations, the author regrets being unable to cite many outstanding publications in this field. Thanks to Y. M. Rangel for critical reading and useful feedback. The author was supported by grants from the National Heart, Lung and Blood Institute, and the National Institute of Neurological Disorders and Stroke, National Institutes of Health.

Competing interests statementThe author declares no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBCL2 | BDNF | CREB | HIF1α | IL-10 | NF-κB | TGFβ | VEGF

FURTHER INFORMATIONAuthor’s homepage: http://neurosurgery.wustl.edu/giddayparklab.htm

SUPPLEMENTARY INFORMATIONSee online article: S1 (table) | S2 (table)Access to this links box is available online.

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cerebral preconditioning and ischaemic tolerance

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